High resolution structures of proteins derived from X-ray crystallography and nuclear magnetic resonance (NMR) provide an invaluable starting-point for our understanding of protein function. However, these structures are most often determined under highly restricted conditions (e.g., solid state or high concentration solutions) and often use highly tailored protein constructs (e.g., deleted loops and hydrophobic regions). Some of our most important structure-function information stems from results obtained when various spectroscopic methods (e.g., fluorescence or circular dichroism) were used to link the high resolution structures to structures prevailing under diverse environments (e.g., low concentration solutions; salts; denaturants; ligands). The rates at which hydrogen located at peptide amide linkages are replaced with deuterium when proteins are incubated in D2O provide the basis for another important method for detecting structural changes in proteins exposed to diverse environments. Recent coupling of amide hydrogen exchange (HX) and mass spectrometry (MS) has placed this method on a cusp of advancement." Advancing HX MS methodology and its application to important areas of biomedical research are the principal long-term goals of research proposed in this application. To achieve these long-term goals, research projects in four areas have been proposed. HX MS methods developed during the current funding period will be used to identify the rules governing folding and unfolding of large proteins and to investigate the structure and formation of amyloid plaques. In a new line of research, HX MS methods will be evaluated for determining equilibrium protein-ligand binding constants and dissociation rate constants. Having established feasibility to detect structural changes in a simple plant viral capsid by HX MS during the current funding period, attention will be directed to detecting structural changes in mammalian picornavirus capsids induced by binding of antiviral agents. The utility of HX MS for investigating the structural basis through which domains in large, multi-domain proteins communicate will be determined. This project will focus on hematopoietic cell kinase (Hck), which is strongly activated in vivo by the HIV Nef protein.